Detection of CBP rearrangements in acute myelogenous leukemia with t(8;16).

The CREB-binding protein (CBP) is a large nuclear protein that regulates many signal transduction pathways and is involved in chromatin-mediated transcription. The translocation t(8;16)(p11;p13.3) consistently disrupts two genes: the CBP gene on chromosome band 16p13.3 and the MOZ gene on chromosome band 8p11. Although a fusion of these two genes as a result of the translocation is expected, attempts at detecting the fusion transcript by reverse transcriptase polymerase chain reaction (RT-PCR) have proven difficult; to date, only one in-frame CBP/MOZ fusion transcript has been reported. We therefore sought other reliable means of detecting CBP rearrangements. We applied fluorescence in situ hybridization (FISH) and Southern blot analyses to a series of AML patients with a t(8;16) and detected DNA rearrangements of both the CBP and the MOZ loci in all cases tested. All six cases examined for CBP rearrangements have breakpoints within a 13 kb breakpoint cluster region at the 5' end of the CBP gene. Additionally, we used a MOZ cDNA probe to construct a surrounding cosmid contig and detect DNA rearrangements in three t(8;16) cases, all of which display rearrangements within a 6 kb genomic fragment of the MOZ gene. We have thus developed a series of cosmid probes that consistently detect the disruption of the CBP gene in t(8;16) patients. These clones could potentially be used to screen other cancer-associated or congenital translocations involving chromosome band 16p13.3 as well.

Study of chromosome rearrangements associated with the trpE26 mutation of Bacillus subtilis.

Chromosome rearrangements involved in the formation of merodiploid strains in the Bacillus subtilis 168-166 system were explained by postulating the existence of intrachromosomal homology regions. This working hypothesis was tested by analysing sequences and restriction patterns of the, as yet uncharacterized, junctions between chromosome segments undergoing rearrangements in parent, 168 trpC2 and 166 trpE26, as well as in derived merodiploid strains. Identification, at the Ia/Ib chromosome junction of both parent strains, of a 1.3 kb segment nearly identical to a segment of prophage SPbeta established the existence of one of the postulated homology sequences. Inspection of relevant junctions revealed that a set of different homology regions, derived from prophage SPbeta, plays a key role in the formation of so-called trpE30, trpE30+, as well as of new class I merodiploids. Analysis of junctions involved in the transfer of the trpE26 mutation, i.e. simultaneous translocation of chromosome segment C and rotation of the terminal relative to the origin moiety of the chromosome, did not confirm the presence of any sequence suitable for homologous recombination. We propose a model involving simultaneous introduction of four donor DNA molecules, each comprising a different relevant junction, and their pairing with the junction regions of the recipient chromosome. The resolution of this structure, resting on homologous recombination, would confer the donor chromosome structure to the recipient, achieving some kind of 'transstamping'. In addition, a rather regular pattern of inverse and direct short sequence repeats in regions flanking the breaking points could be correlated with the initial, X-ray-induced, rearrangement.

The genomic distribution of intraspecific and interspecific sequence divergence of human segmental duplications relative to human/chimpanzee chromosomal rearrangements

Background: It has been suggested that chromosomal rearrangements harbor the molecular footprint of the biological phenomena which they induce, in the form, for instance, of changes in the sequence divergence rates of linked genes. So far, all the studies of these potential associations have focused on the relationship between structural changes and the rates of evolution of single-copy DNA and have tried to exclude segmental duplications (SDs). This is paradoxical, since SDs are one of the primary forces driving the evolution of structure and function in our genomes and have been linked not only with novel genes acquiring new functions, but also with overall higher DNA sequence divergence and major chromosomal rearrangements.Results: Here we take the opposite view and focus on SDs. We analyze several of the features of SDs, including the rates of intraspecific divergence between paralogous copies of human SDs and of interspecific divergence between human SDs and chimpanzee DNA. We study how divergence measures relate to chromosomal rearrangements, while considering other factors that affect evolutionary rates in single copy DNA. Conclusion: We find that interspecific SD divergence behaves similarly to divergence of single-copy DNA. In contrast, old and recent paralogous copies of SDs do present different patterns of intraspecific divergence. Also, we show that some relatively recent SDs accumulate in regions that carry inversions in sister lineages.

Theoretical study of catalytic reactions of carbenes: haptotropic rearrangements and the Dötz reaction

En aquesta tesi s'han estudiat mecanismes de reaccions de cicloanulació en carbens de Fischer a través de mètodes teòrics, concretament fent servir el nivell de teoria B3LYP/(Wachters' basis / 6-31G**). Els alcoxi- i amino carbens de pentacarbonil crom, ja siguin vinílics o aromàtics, reaccionen amb acetilè per produir fenols, naftols o derivats ciclopentadiè o indè substituïts amb el Cr(CO)3 coordinat, d'una manera regioselectiva. L'objectiu d'aquest treball és discutir ambudes reaccions competitives particularment a la reacció de Dötz, la qual durant els darrers anys ha estat explorada experimentalment per W.D. Wulff, C.P. Casey, R. Aumann i J. Barluenga entre altres diferents propostes mecanístiques. A més K.H. Dötz va demostrar que la coordinació del Cr(CO)3, un cop l'anell ja està format, pot patir canvis haptotròpics, és a dir, la caminada del complex metàl·lic d'un anell a un altre -generalment almenys substituït- canviant la seva hapticitat (coordinació pi amb els membres de l'anell). Llavors, s'han estudiat les migracions haptotròpiques intramoleculars en petits hidrocarbons aromàtics policíclics amb l'objectiu d'analitzar les rutes de reacció per les quals aquestes reaccions es porten a terme

Sigmatropic rearrangements of 'onium' ylids

Rearrangement reactions occupy a special place within the canon of organic synthesis, by virtue of the inherently high efficiency of chemical processes which form and breyak bonds by redistribution of electrons around a retained atomic framework. Within the broader class, sigmatropic rearrangements are chemical processes defined by mechanisms involving unimolecular migration of sigma-bonds with concomitant redistribution of one or more pi-bonds. Sigmatropic processes may involve uncharged or charged species, with the charges located on carbon or heteroatoms; the latter reaction type is the subject of this tutorial review.

Asymmetric ammonium ylid rearrangements: the effect of nitrogen asymmetry

[2,3]-Sigmatropic rearrangements of allylic ammonium ylids derived from glycinoylcamphorsultams are highly selective in terms of relative and absolute stereocontrol only when acyclic alkenes are present. When chiral esters of ylids derived from N-methyltetrahydro-pyridine ('NMTP') undergo rearrangement, the reactions show exclusive cis-stereoselectivity but the products are obtained with virtually no absolute stereocontrol. These observations support the notion that sigmatropic rearrangements of N-chiral ammonium ylids are controlled by nitrogen stereogenicity. (c) 2006 Elsevier Ltd. All rights reserved.

Regioselective Beckmann rearrangements of furanoside and pyranoside-derived oximes

The Beckmann rearrangement is a useful reaction employed to provide access to amides from oxime substrates. Applied to cyclic structures, the Beckmann rearrangement leads to ring expansion and allows access to cyclic lactams. Our investigations focused upon the synthesis of glycoside-derived lactams from oxime precursors. In probing a range of conditions, we observed that 2,4,6-trichloro[1,3,5]triazine (TCT) was an effective and mild promoter of the rearrangement affording pyrano- and heptanoside lactam products with excellent regioselectivities.

Probing the effect of allylic substitution on cyclic ammonium ylid rearrangements

The [2,3]-sigmatropic rearrangement of tetrahydropyridine-derived ammonium ylids is a valuable method for the preparation of substituted pyrrolidine carboxylates. The presence of an allylic substituent does not intrinsically reduce the yield of rearrangements, and the diastereoselectivity of rearrangement is related to the structure of the diazo reactant. The method represents a very rapid means of accessing complex pyrrolidines, as shown by preparation of a precursor to the core of lactacystin.

Comparative genomics of Brachyspira pilosicoli strains: genome rearrangements, reductions and correlation of genetic compliment with phenotypic diversity.

Background. The anaerobic spirochaete Brachyspira pilosicoli causes enteric disease in avian, porcine and human hosts, amongst others. To date, the only available genome sequence of B. pilosicoli is that of strain 95/1000, a porcine isolate. In the first intra-species genome comparison within the Brachyspira genus, we report the whole genome sequence of B. pilosicoli B2904, an avian isolate, the incomplete genome sequence of B. pilosicoli WesB, a human isolate, and the comparisons with B. pilosicoli 95/1000. We also draw on incomplete genome sequences from three other Brachyspira species. Finally we report the first application of the high-throughput Biolog phenotype screening tool on the B. pilosicoli strains for detailed comparisons between genotype and phenotype. Results. Feature and sequence genome comparisons revealed a high degree of similarity between the three B. pilosicoli strains, although the genomes of B2904 and WesB were larger than that of 95/1000 (~2,765, 2.890 and 2.596 Mb, respectively). Genome rearrangements were observed which correlated largely with the positions of mobile genetic elements. Through comparison of the B2904 and WesB genomes with the 95/1000 genome, features that we propose are non-essential due to their absence from 95/1000 include a peptidase, glycine reductase complex components and transposases. Novel bacteriophages were detected in the newly-sequenced genomes, which appeared to have involvement in intra- and inter-species horizontal gene transfer. Phenotypic differences predicted from genome analysis, such as the lack of genes for glucuronate catabolism in 95/1000, were confirmed by phenotyping. Conclusions. The availability of multiple B. pilosicoli genome sequences has allowed us to demonstrate the substantial genomic variation that exists between these strains, and provides an insight into genetic events that are shaping the species. In addition, phenotype screening allowed determination of how genotypic differences translated to phenotype. Further application of such comparisons will improve understanding of the metabolic capabilities of Brachyspira species.

A modular flow reactor for performing Curtius rearrangements as a continuous flow process

The use of a mesofluidic flow reactor is described for performing Curtius rearrangement reactions of carboxylic acids in the presence of diphenylphosphoryl azide and trapping of the intermediate isocyanates with various nucleophiles.

Complex rearrangements in patients with duplications of MECP2 can occur by fork stalling and template switching

Duplication at the Xq28 band including the MECP2 gene is one of the most common genomic rearrangements identified in neurodevelopmentally delayed males. Such duplications are non-recurrent and can be generated by a non-homologous end joining (NHEJ) mechanism. We investigated the potential mechanisms for MECP2 duplication and examined whether genomic architectural features may play a role in their origin using a custom designed 4-Mb tiling-path oligonucleotide array CGH assay. Each of the 30 patients analyzed showed a unique duplication varying in size from similar to 250 kb to similar to 2.6 Mb. Interestingly, in 77% of these non-recurrent duplications, the distal breakpoints grouped within a 215 kb genomic interval, located 47 kb telomeric to the MECP2 gene. The genomic architecture of this region contains both direct and inverted low-copy repeat (LCR) sequences; this same region undergoes polymorphic structural variation in the general population. Array CGH revealed complex rearrangements in eight patients; in six patients the duplication contained an embedded triplicated segment, and in the other two, stretches of non-duplicated sequences occurred within the duplicated region. Breakpoint junction sequencing was achieved in four duplications and identified an inversion in one patient, demonstrating further complexity. We propose that the presence of LCRs in the vicinity of the MECP2 gene may generate an unstable DNA structure that can induce DNA strand lesions, such as a collapsed fork, and facilitate a Fork Stalling and Template Switching event producing the complex rearrangements involving MECP2.

Variability of 18S rDNA locus among Symphysodon fishes: chromosomal rearrangements

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Assignment of chromosome rearrangements between X chromosomes of human and cattle by laser microdissection and Zoo-FISH

Cross-species fluorescence in-situ hybridization (Zoo-FISH) was performed on cattle metaphase spreads using Homo sapiens X chromosome (HSAX) painting probes specific for the p- and q-arms to identify the cytogenetic location of a chromosome breakpoint between HSAX and the Bos taurus X chromosome (BTAX). The existence of a breakpoint is strongly suggested by recent radiation hybrid and FISH mapping results. Hybridization probes were generated by microdissection of HSAX p- and q-arms using the contact-free technology of Laser Microdissection and Pressure Catapulting (LMPC), amplification of the isolated chromosome material by DOP-PCR, and labelling of the PCR products with digoxigenin in a secondary PCR. Independent Zoo-FISH of the two painting probes on bovine metaphase chromosomes (detected by antidigoxigenin-fluorescein) resulted in clear hybridization signals on BTAX. A breakpoint was identified between HSAXp and HSAXq on BTAX, and narrowed down between the G-bands BTAXq25 and BTAXq26. The assumed centromere transposition between HSAX and BTAX associated with the rearranged chromosome segments is supported by cytogenetic assignments of the genes BGN and G6PD to BTAX.